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Simultaneous measurements of urinary free cortisol and cortisone for the assessment of functional glucocorticoid activity.

To the Editor:

Urinary free cortisol (UFF) excretion in 24-h urine samples is frequently measured in clinical laboratories to assess functional glucocorticoid activity (fGcA). The 24-h UFF excretion serves as an integrated measure of blood free cortisol concentrations during the entire day (1). Increasing evidence suggests that simultaneous measurements of more than 1 analyte in the same sample material will bring more confidence to the acceptance or rejection of specific diagnoses (2-4). Accordingly, clear advantages have been reported regarding the identification of hypercortisolism, e.g., in patients with confirmed Cushing syndrome (2) and type1 diabetes mellitus (4), with simultaneous measurements of UFF and urinary free cortisone (UFE) compared with UFF alone. Variations in activity of 11[beta]-hydroxysteroid dehydrogenase type2, a highly expressed enzyme in the human kidney that inactivates cortisol to cortisone, can confound renal cortisol output (UFF). Accordingly, the usual renal 24-h UFE excretion is about twice that of UFF (2-4).

Recent examinations have shown a correlation coefficient of 0.71 for the relation of 24-h plasma free cortisol and cortisol production rate divided by body surface area, suggesting that only approximately 50% of the variation of plasma free cortisol can be explained by cortisol production (5). This result implies that additional relevant (endocrine, metabolic, nutritional) influences on bioavailable plasma cortisol exist apart from the adrenal gland's glucocorticoid secretion, and it led us to examine whether the combined measurement of UFF + UFE may show better associations with such additional influences than does UFF alone.

For this theory, we studied 30 healthy lean adults (22-44 years old; body mass index 20-25 kg/[m.sup.2], 15 women) and measured their 24-h urinary excretion rates of UFF, UFE, tetrahydrocortisol (THF), 5a-tetrahydrocortisol (5a-THF), and tetrahydrocortisone (THE) (4). The sum of the latter 3 major urinary glucocorticoid metabolites (GC3), which reflects adrenocortical glucocorticoid secretion, was determined to adjust for the adrenal gland's secretory activity. The ratio of 5a-THF:THF served to assess activity of the cortisol-catabolizing enzyme 5a-reductase relative to 5[beta]-reductase. Measurement of 24-h total nitrogen excretion provided an index for protein intake. Circulating leptin, known to interfere with the glucocorticoid status, was measured together with total plasma cortisol in venous blood samples.

As expected in healthy adults with statistically indistinguishable body mass index values [mean (SD): 22.8 (1.7) for men and 21.9 (1.8) kg/[m.sup.2] for women], GC3 excretion was higher in men than women [10.1 (3.2) and 6.9 (2.3) mg/day, respectively, P <0.01], as was 5a-reductase [1.3 (0.5) and 0.9 (0.4), P <0.05], whereas plasma leptin was lower in men than in women [2.8 (1.6) and 7.6 (4.9) [micro]g/L, respectively, P <0.01]. Plasma cortisol [178 (53) and 245 (164) [micro]g/L] and UFF + UFE [127 (49) and 120 (51) [micro]g/day] did not significantly differ between the sexes, nor did UFF [44.8 (17.4) and 38.3 (19.0) [micro]g/day] or nitrogen excretion [904 (246) and 821 (257) mmol/day].

Two separate regression models were run for each sex, one with the conventional outcome (dependent variable) UFF alone, and the other with the potential bioactive free glucocorticoids UFF + UFE as an outcome. In both models, the only significant associations observable for men were between GC3 and UFF ([beta], 3.7; P <0.01) and GC3 and UFF + UFE ([beta], 10.3; P <0.01). In women, in addition to a priori adjusted GC3, nitrogen also showed an association with UFF, resulting in a total explained variation of 47% (Table 1) for UFF alone. However, with UFF + UFE as the outcome, total [R.sup.2] in women increased to 0.72 and--in addition to urinary nitrogen--plasma leptin also explained a significant portion of variation of potential fGcA after adjustment for glucocorticoid secretion (GC3; Table 1). In line with the known stimulating effect of increased 5a-reductase activity on cortisol clearance, a trend (P = 0.057) for a negative association of this enzyme's activity index (5a-THF/THF) with UFF + UFE was seen (Table 1). Accordingly, metabolic and nutritional influences on fGcA (assessed in 24-h urine samples) can be unraveled if the influence of the adrenocortical secretory activity is taken into account (e.g., as GC3).

With both UFF + UFE and UFF (the conventional measure for fGcA), increases in the protein intake-related postmeal plasma cortisol, as reported in the literature, are mirrored by the significant positive [beta] values for 24-h nitrogen excretion rates. However, the frequently reported interaction of leptin with the hypothalamus-pituitary-adrenal axis and the 5a-reductase influence on cortisol clearance remain masked if only UFF is analyzed. A major limitation of our study is that no individuals with abnormal glucocorticoid values were examined. Although recent findings (2, 4) and the present data suggest that UFE may be a useful complementary analyte to UFF for a more meaningful assessment of fGcA, further studies on healthy individuals and hypercortisolemic patients are required before manufacturers can be encouraged to develop adequate multiple glucocorticoid metabolite immunoassays for clinical use.

Grant/funding support: None declared. Financial disclosures: None declared.

DOI: 10.1373/clinchem.2007.092031


(1.) Orth DN. Cushing's syndrome. N Engl J Med 1995;332:791-803.

(2.) Lin CL, Wu TJ, Machacek DA, Jiang NS, Kao PC. Urinary free cortisol and cortisone determined by high performance liquid chromatography in the diagnosis of Cushing's syndrome. J Clin Endocrinol Metab 1997;82:151-5.

(3.) Taylor RL, Machacek D, Singh RJ. Validation of a high-throughput liquid chromatography-tandem mass spectrometry method for urinary cortisol and cortisone. Clin Chem 2002;48:1511-9.

(4.) Remer T, Maser-Gluth C, Boye KR, Hartmann MF, Heinze E, Wudy SA. Exaggerated adrenarche and altered cortisol metabolism in Type 1 diabetic children. Steroids 2006;71:591-8.

(5.) Purnell JQ, Brandon DD, Isabelle LM, Loriaux DL, Samuels MH. Association of 24-hour cortisol production rates, cortisol-binding globulin, and plasma-free cortisol levels with body composition, leptin levels, and aging in adult men and women. J Clin Endocrinol Metab 2004;89: 281-7.

Thomas Remer [1] * Christiane Maser-G1uth [2]

[1] Research Institute of Child Nutrition Dortmund, Germany

[2] University of Heidelberg Department of Pharmacology Heidelberg, Germany

* Address correspondence to this author at: Forschungsinstitut fur Kinderernahrung, Research Institute of Child Nutrition, Department of Nutrition and Health, Heinstuck 11, 44225 Dortmund, Germany. Fax 49-231-711581; e-mail
Table 1. Predictors of UFF and potential bioactive free
glucocorticoids (UFF + UFE) in healthy women (n = 15). (a)

Models Predictors P R2

UFF GC3 3.3 0.078 0.11
 Urinary nitrogen 0.05 0.006 0.36
UFF + UFE GC3 15.5 0.001 0.21
 Urinary nitrogen 0.11 0.003 0.29
 Plasma leptin -7.3 0.001 0.22
 5a-THF/THF -35.7 0.057 0.09
 0.72 (b)

(a) Stepwise multiple regression results
(models a priori adjusted for GC3).

(b) Total R2for significant predictors (P < 0.05)
without 5a-reductase index (5a-THF/THF).
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Title Annotation:Letters
Author:Remer, Thomas; Maser-G1uth, Christiane
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Oct 1, 2007
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